Amniotic fluid formulation for treatment of joint pain or disorders

ABSTRACT

A human amniotic fluid formulation has been developed for administration into a joint or associated soft tissue such as a tendon or ligament for treatment of pain, degeneration, or injury. The formulation is a sterile de-cellularized human amniotic fluid (D-HAF), devoid of amniotic stem cells and elements of micronized membrane or chorion particles, which has not been heat treated or treated with ethidium bromide. The formulation is optionally diluted, or concentrated, depending on the severity of the disorder or injury. Examples demonstrate efficacy in treatment of pain, disease, disorder, degeneration or injury of a joint or associated soft tissues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/912,096, filedMar. 5, 2018, which application is a continuation-in-part of U.S. Ser.No. 15/289,367, filed Oct. 10, 2016, now U.S. Pat. No. 9,907,821, issuedMar. 6, 2018, which claims priority to U.S. Provisional Application No.62/239,713, filed Oct. 9, 2015, each of which is incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made without government support or Grants awarded bythe National Institutes of Health. The government has no rights in theinvention.

FIELD OF THE INVENTION

This generally relates to the treatment, prevention or alleviation ofjoint pain, disease, injury, disorder, or degeneration by administrationof a formulated human amniotic fluid solution.

BACKGROUND OF THE INVENTION

Orthopaedic ailments and movement disorders have a direct causal impacton a person's ability to perform basic functions such as walking,reading, driving and other common activities. Good health contributessignificantly to a person's quality of life and their ability to performcommon daily activities and simple functions in their routineenvironments. Orthopaedic and movement disorders and diseases can beseverely incapacitating and occur in a wide variety of forms. Millionsof people suffer from some form of movement ailment. As an example,torticollis, a condition in which the head becomes persistently turnedto one side, is often associated with painful muscle spasms. The livesof individuals with this disorder are negatively impacted due toconsistent pain and limited movement. Treatment for torticollis istargeted to relax the contracted neck muscles involved. Treatmentsinclude medication, physical devices, botulinum toxin, physical therapy,stretching exercises, and surgery. In most people, torticollis resolvesin several days to a few weeks. A few people will develop continuingneck problems for months to years.

Acute and chronic pain, loss of structural function and range ofmovement can occur due to disease, injury, disorders (such asmalalignment), or degeneration. There are two principal types of tissueinvolved: bony tissues, including joints and the collagenous surfacesthereon, and soft tissues such as ligaments, tendons, and capsules.

Treatment of degenerative joint disease (osteoarthritis) is discussed inrelationship to pathogenesis of five clinical entities: 1) occurring inhigh motion joints and associated with synovitis; 2) associated with lowmotion joints; 3) “non-progressive” articular cartilage erosion; 4)secondary to other identified problems (intra-articular fractures,ligamentous damage, wounds, septic arthritis, osteochondrosis); and 5)chondromalacia of the patella. In addition to direct damage to articularcartilage, synovitis and capsulitis and depletion of matricalglycosaminoglycans and proteoglycans play important roles.

The natural healing response in damaged articular cartilage isinadequate. The principles of treatment are divided into: 1) preventionor treatment of primary causes. These include the effects of tracksurface, shoeing, and the appropriate resolution of intra-articularfractures, septic arthritis and osteochondritis dissecans. 2) treatmentof active soft tissue disease contributing to articular cartilagedegeneration, including rest, physical therapy, synovectomy andadministration of anti-inflammatory drugs, sodium hyaluronate andpolysulfated glycosaminoglycans. 3) treatment of articular cartilageloss or degeneration, including articular cartilage curettage,subchondral bone drilling, osteophyte removal, articular cartilagegrafting and arthrodesis.

In sum, methods of treatment are limited to surgical procedures,palliative (injection of steroids or anesthetics), and injection of“repair promoters” such as hyaluronic acid, none of which are totallyeffective.

It is an object of the present invention to provide a formulation thatcan be used for the management of chronic joint pain, injury ordisorders.

It is a further object of the present invention to provide a formulationthat induces healing as well as increases joint mobility and decreasespain.

SUMMARY OF THE INVENTION

A human amniotic fluid formulation has been developed for treatment ofjoint disease, injury, pain or degeneration. The formulation is asterile de-cellularized human amniotic fluid (D-HAF), typicallyadministered by injection. D-HAF contains over 300 human growth factors.D-HAF is devoid of amniotic stem cells and elements of micronizedmembrane or chorion particles. The formulation may be diluted prior toadministration, based on the severity of the disorder or injury.

D-HAF is prepared from human amniotic fluid from which the amniotic stemcells and particulate matter have been removed. In the preferredembodiment, the process consists of separating the cells from the AFusing centrifugation and then filtering the supernatant through a seriesof filtration devices to remove all remaining cells and bioburden. Eachlot is tested for bioburden and is certified sterile to contain <1harmful organisms. The purified fluid is sterilized without the use ofharsh terminal irradiation, e-beam or Ethylene Oxide (EO).

Methods for treating pain, disease, disorder, degeneration or injury ofa joint or associated soft tissue using the D-HAF formulation aredescribed. In some embodiments, the formulation is administered to treatjoint conditions such as joints with pain, injury, degeneration,cartilage wear or tear, joints that have been fractured, arthritis,bursitis, dislocations, tendinitis, and tenosynovitis. In someembodiments, the formulation is administered to bones that have hadprosthetic implants, pins, screws or plates attached or implanted intothem, to promote healing and repair, and to reduce inflammation. In someembodiments, injections are administered to assist in resurfacing andrepair or regeneration of cartilage. In some embodiments, theformulation is administered to assist in soft tissue repair, such asrepair of tom or strained ligaments or tendons. In one embodiment, theformulation is administered by injection to joints, bursae, tendonsheaths, or soft tissues at/near the site of injury. In someembodiments, the formulation is injected into and/or adjacent to thejoint to be treated, on an as-needed basis. In another embodiment, theformulation is sprayed onto, soaked into, or powder dispersed onto theimplant or prosthetic. This can include matrices, implants and sutures.

Methods for treating pain, disease, disorder, degeneration or injury ofa joint or associated soft tissue using the described formulation incombination with one or more therapeutic, prophylactic or diagnosticagents are also described. In some embodiments, antimicrobial agents,analgesic agents, local anesthetic agents, anti-inflammatory agents,immunosuppressant agents, anti-allergenic agents, enzyme cofactors,essential nutrients, growth factors, and/or combinations thereof areco-administered.

A kit containing one or more single, sterile units of D-HAF in fluid, orsolid form, and instructions on how the dose is to be used for treatmentof pain, disease, disorder, degeneration or injury of a joint orassociated soft tissue is also developed. The kit can also include aunit dose of sterile water, saline or buffer for dilution. The kit isgenerally used by practitioners for patients with pain, disease,disorder, degeneration or injury of a joint or associated soft tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a potential mechanism of action of the steriledecellularized human amniotic fluid for regeneration of tissues andcells in the body.

FIG. 2 is bar graph showing patients with validated pain assessmentscores ranging from I to 10 (10 being the worst) at pre-treatment, and3-month,6-month, and 12-month post treatment intervals. Percentagesindicate the percentage of improvement relative to pre-treatment scores.

FIGS. 3A-3C are graphs for quantifying Wnt-4 protein in the steriledecellularized human amniotic fluid (D-HAF); FIG. 3A is a linearregression curve and FIG. 3B a log-log regression curve based on a knownstandard; and FIG. 3C is a bar graph showing the quantity of Wnt-4 inD-HAF. LOD: limit of detection.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

“Active Agent,” as used herein, refers to a physiologically orpharmacologically active substance that acts locally and/or systemicallyin the body. An active agent is a substance that is administered to apatient for the treatment (e.g., therapeutic agent), prevention (e.g.,prophylactic agent), or diagnosis (e.g., diagnostic agent) of a diseaseor disorder.

“Effective amount” or “therapeutically effective amount,” refers to anamount of the D-HAF effective to alleviate, delay onset of, or preventone or more symptoms, particularly of a disease or disorder of a joint.“Control” refers to an experiment performed without the activeingredient, for example without sterile DHAF composition. In otherexamples, controls are ones receiving only tissue grafts, or implantswithout the DHAF composition. Controls are known in the art.

“Enhance”, “increase”, “stimulate”, “promote”, “decrease”, “inhibit” or“reduce” are used relative to a control. Controls are known in the art.For example, an increase response in a subject or cell treated with acompound is compared to a response in subject or cell that is nottreated with the compound.

“Growth factors” generally refer to a group of proteins or hormones thatstimulate the cellular growth. Growth factors play an important role inpromoting cellular differentiation and cell division, and they occur ina wide range of organisms.

“Pharmaceutically acceptable,” refers to compounds, carriers,excipients, compositions, and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

“Biocompatible” and “biologically compatible,” generally refer tomaterials that are, along with any metabolites or degradation productsthereof, generally non-toxic to the recipient, and do not cause anysignificant adverse effects to the recipient. Generally speaking,biocompatible materials are materials which do not elicit a significantinflammatory or immune response when administered to a patient.

“Biodegradable Polymer,” as used herein, generally refers to a polymerthat will degrade or erode by enzymatic action and/or hydrolysis underphysiologic conditions to smaller units or chemical species that arecapable of being metabolized, eliminated, or excreted by the subject.The degradation time is a function of polymer composition, morphology,such as porosity, particle dimensions, and environment.

“Nanoparticle,” as used herein, generally refers to a particle having adiameter, such as an average diameter, from about 10 nm up to but notincluding about 1 micron, preferably from 100 nm to about 1 micron. Theparticles can have any shape. Nanoparticles having a spherical shape aregenerally referred to as “nanospheres”.

“Microparticle,” as used herein, generally refers to a particle having adiameter, such as an average diameter, from about 1 micron to about 100microns, preferably from about 1 micron to about 50 microns, morepreferably from about 1 to about 30 microns. The microparticles can haveany shape. Microparticles having a spherical shape are generallyreferred to as “microspheres”.

“Molecular weight,” as used herein, generally refers to the relativeaverage chain length of the bulk polymer, unless otherwise specified. Inpractice, molecular weight can be estimated or characterized usingvarious methods including gel permeation chromatography (GPC) orcapillary viscometry. GPC molecular weights are reported as theweight-average molecular weight (Mw) as opposed to the number-averagemolecular weight (Mn). Capillary viscometry provides estimates ofmolecular weight as the inherent viscosity determined from a dilutepolymer solution using a particular set of concentration, temperature,and solvent conditions.

“Mean particle size,” as used herein, generally refers to thestatistical mean particle size (diameter) of the particles in apopulation of particles. The diameter of an essentially sphericalparticle may refer to the physical or hydrodynamic diameter. Thediameter of a non-spherical particle may refer preferentially to thehydrodynamic diameter. As used herein, the diameter of a non-sphericalparticle may refer to the largest linear distance between two points onthe surface of the particle. Mean particle size can be measured usingmethods known in the art, such as dynamic light scattering.

“Implant,” as generally used herein, refers to a polymeric device orelement that is structured, sized, or otherwise configured to beimplanted, preferably by injection or surgical implantation, in aspecific region of the body so as to provide therapeutic benefit byreleasing one or more therapeutic, prophylactic or diagnostic agentsover an extended period of time at the site of implantation.

“Hyaline cartilage” refers to the connective tissue covering the jointsurface. By way of example only, hyaline cartilage includes, but is notlimited to, articular cartilage, costal cartilage, and nose cartilage.

“Connective tissue” refers to any tissue that connects and supportsother tissues or organs, and includes but is not limited to a ligament,a cartilage, a tendon, a bone, and a synovium of a mammalian host.

The term “in combination” refers to the use of more than one therapeuticagent. The use of the term “in combination” does not restrict the orderin which said therapeutic agents are administered to a subject.

II. Compositions

Formulations of purified human amniotic fluid are provided. Typically,the formulations include diluted sterile de-cellularized human amnioticfluid (D-HAF), either in fluid form or solid form, for example,lyophilized powder, aloneor in combination with appropriate excipients.Other active agents can be included. D-HAF contains over 300 humangrowth factors. D-HAF is devoid of amniotic stem cells and elements ofmicronized membrane or chorion particles.

A. Purified Amniotic Fluid Formulation

Amniotic fluid (“AF”) contains nutrients and growth factors thatfacilitate fetal growth, provides mechanical cushioning andantimicrobial effectors that protect the fetus, and allows assessment offetal maturity and disease. AF typically contains mixtures of growthfactors, pro- inflammatory cytokines and anti-inflammatory cytokines, aswell as a variety of macromolecules including carbohydrates, proteinsand peptides, lipids, lactate, pyruvate, electrolytes, enzymes, andhormones.

In the preferred embodiment, the raw fluid directly collected from thesource is not heat-treated, chemical-treated, fractionated to producethe disclosed formulations. In some embodiments, the formulation retainsmore than 50%, more than 60%, more than 70%, more than 80%, orpreferably more than 90%, of the amniotic factors present in the rawfluid. In some embodiments, the formulations are not diluted with anyadditional solution. In other embodiments, the formulations are notconcentrated relative to the raw fluid.

In some embodiments, the formulation is a diluted sterilede-cellularized human amniotic fluid (D-HAF). D-HAF contains over 300human growth factors. D-HAF is devoid of amniotic stem cells andelements of micronized membrane or chorion particles. The purified fluidis sterilized without the use of harsh terminal irradiation, e-beam orEthylene Oxide (EO). In the preferred embodiment, the process consistsof separating the stem cells from the AF using centrifugation andutilizing a series of filtration devices to remove all remaining cellsand bioburden. Each lot is tested for bioburden and is certified sterileto contain <1 harmful organism.

Generally, methods of preparing sterile de-cellularized amniotic fluidsinvolve a series of centrifugation and filtrations steps. Preferredmethods of preparing sterile de-cellularized amniotic fluid aredescribed in detail in U.S. application Ser. No. 15/053,497.

1. Method of Obtaining Amniotic Fluid

In some embodiments, the amniotic fluid is collected in a sterileoperating room environment during an elective C-section. Typically, theprocedure is performed using an ultrasound device to provide guidancefor the process of obtaining human fluid from the woman, inserting ablunt tip needle into the amniotic sac of the woman, attaching the blunttip needle to a three-way stopcock, connecting a Luer lock syringe tothe three-way stopcock, connecting a first end of a length of steriletubing with the three-way stopcock, and collecting sterilely theamniotic fluid through the blunt tip needle and sterile tubing into acollection container.

In this embodiment, the sterile collection container can include a pumpwith a suction device, typically a low suction device or a spring loadedlow suction device. The suction device is fluidly connected to aninternal balloon. This embodiment further includes manually pumping upthe internal balloon in the sterile collection container using the lowsuction device to allow a low-level suction and collection of theamniotic fluid.

In one embodiment, tools to obtain sterilely filtered human amnioticfluid from a woman, include a three-way stopcock, a sterile blunt tipneedle aseptically attached to the three-way stopcock, a luer locksyringe aseptically connected to the three-way stopcock, a steriletubing aseptically connected to the three-way stopcock, a collectioncontainer or a collection container including a pump with suction deviceconnected with the sterile tubing, a set of filters having the pore sizeof about 5 μm to about 10 μm, a set of capsule or cartridge filtershaving the pore size of about 1 μm, a set of capsule or cartridgefilters having the pore size of about 0.45 μm or 0.2 μm, a set ofsterile syringes or vials to store the sterile filtered amniotic fluidand, optionally, operating instructions on using the kit to obtainsterilely filtered human amniotic fluid. The filters having the poresize of from about 5 μm to about 10 μm and the capsule or cartridgefilters can be made from cellulose ester, glass fiber or nylon.

The sterile collection container may include a pump with a suctiondevice, which may be a low suction device or spring loaded low suctiondevice. The suction device may be fluidly connected to an internalballoon. The internal balloon may be pumped up in the sterile collectioncontainer using the low suction device to allow a low-level suction andcollection of the amniotic fluid. The sterile collection container mayinclude a vent having a cap.

In one embodiment, utilizing the incision site immediately prior toperforming the C-section and with ultrasound guidance to protect thefetus and mother provides a minimal or no risk environment forcollection. Collection is achieved via a low level suction establishedwithin a collection container and/or via gravity. Typically, after highspeed centrifugation filtration with 5 to 10 μm filters (low proteinbinding filter) is used to complete the removal of cells and largeparticles. Submicron filtration is then conducted with 1 μm and 0.45 μmor/and 0.2 μm filters (low protein binding filter), two in a seriesconnection, to remove gross contaminates. Under this condition, solublegrowth factors will pass through this filter to achieve a semi-sterilecondition, very low bioburden counts. If under a strict asepticoperation condition, a 10³ sterility assurance level is achieved. A 10⁶sterility assurance level can be achieved by submicron filtration with a0.22 μm filter (low protein binding filter) into sterile packaging toachieve a sterile product. The filtrate is monitored after eachfiltration step to determine which components were removed and then todetermine which process to use to achieve the desirable product.

Cells, large particles and other undissolvables are removed from thehuman amniotic fluid by centrifuging or depth filtering the humanamniotic fluid. In some embodiments, the human amniotic fluid iscentrifuged at about5,000 rpm to about 10,000 rpm for between about 30minutes and about 60 minutes. In this embodiment filters of about 5 μmto about 10 μm are used for the first filtration. These are typicallycellulose ester filters, glass fiber filters, nylon capsule filters ornylon cartridge filters. Filters with a pore size of 1.0 μm are capsulefilters or cartridge filters, typically formed of poly ether sulfone,poly vinylidene fluoride or cellulose acetate membranes. Filters with apore size of 0.45 μm or 0.2 μm are capsule filters or cartridge filters,typically formed of poly ether sulfone membrane filters, poly vinylidenefluoride or cellulose acetate membranes.

One may use membrane filters including or made of hydrophilicpolyethersulphone (PES) to filter protein solutions. Filter disks forsmall volumes and different sizes of cartridges for larger volumes suchas 1 litre and more. Hydrophobic membranes like PTFE which are designedfor liquids devoid proteins should not be used. Start withcentrifugation at 5000 to 8000 rpm for at least 30 minutes. Next, thesupernatant is filtered with a prefilter to remove residual proteinaggregates and precipitates in suspension (AP20 can be used). If onedirectly uses a 0.6/0.2 μm filter, after prefiltration, one mayexperience slow filtration rates and the flow may stop too quickly. Itmay be desirable to make intermediate filtration steps using 1.2 μm and0.8 μm membranes. Typically, a final filtration through 0.2 μm isnecessary to get the best sterility assurance level and produce asterile amniotic fluid for injections. The final filtrate can be storedin frozen condition at about −20° C. to about −80° C. for long termstorage.

The sterile amniotic fluid can be lyophilized to yield a lyophilizate.The sterilely filtered amniotic fluid may be distributed in vialsequipped with special rubber stoppers for sterile lyophilization. Thelyophilization is carried out in a sterile environment. The rubberstoppers on the vials are then automatically pushed down in the freezedryer to definitively close them. Finally, an aluminum cap is sealed oneach vial to protect its sterile content.

The lyophilizate can be irradiated by e-beam irradiation or gamma rayirradiation to insure the sterility. The lyophilized amniotic fluid maybe stored at +4° C. or room temperature for at least one year withoutdecrease of its biological activity.

The sterile lyophilized amniotic fluid may be reconstituted by addingthe initial volume of sterile water to the powder in order to restore atransparent and homogeneous physiological liquid.

2. Sources of Amniotic Fluid Formulations

The amniotic fluid formulations are prepared from sterile human amnioticfluid obtained from a pregnant woman. Suitable sources, e.g., of humanAF, include AF that is obtained from patients, who are undergoingamniocentesis, patients who are undergoing a Caesarean section delivery,and patients undergoing normal delivery using a specially designedreceptacle to collect the fluid after rupture of membranes.

The de-cellularized human amniotic fluid (D-HAF) formulations can bestored for long periods of time, allowing for a broad range ofapplication methods, including distribution and storage as aerosols,solutions, powders, etc. In some embodiments, the sterile D-HAF isrefrigerated at about 1° C. to about 10° C. for long-term storage. In afurther embodiment, the sterile D-HAF is refrigerated at 4° C. for up to12 months and more. Preferably, the long-term storage does not reducethe quantity of the total soluble proteins or factors present in theD-HAF. For some embodiments, the total soluble proteins retained afterlong-term storage in refrigerated conditions is about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the fresh D-HAF.

D-HAF formulations containing amniotic factors can be supplied as aclear one-part solution in a suitable container for storage at 4° C., orfor storage at −20° C., or at −80° C. For example, liquid formulationsin prefilled aliquots can be suitable for storage at 1-5° C., or forstorage at −20° C., or at −80° C. The liquid formulation can be suitablefor topical application in a nebulizer or an inhaler. In otherembodiments, the fluid can be supplied as a kit that can be stored at 4°C., at −20° C., or at −80° C. until needed.

In some embodiments, D-HAF formulations use a final filtration through0.2 μm is necessary to get the best sterility assurance level andproduce a sterile amniotic fluid without any irradiation. In someembodiments, D-HAF formulations have a 10-6 sterility assurance levelwithout irradiation. In other embodiments, lyophilisate derived fromamniotic fluid through lyophilization may be irradiated by e-beamirradiation or gamma ray irradiation to add another guarantee for thefinal sterility of the powder.

In some embodiments, D-HAF formulations are synthesized amniotic fluidto include all the known amniotic factors for the same therapeutic,and/or prophylactic properties in treating pain, disease, disorder,degeneration or injury of a joint or associated soft tissue.

The sterilely filtered human amniotic fluid contains growth factors. Thegrowth factors include human growth hormone, transforming growth factorbeta 1, vascular endothelial growth factor, epidermal growth factor,transforming growth factor beta 3, and growth differentiation factor 11or combinations thereof.

B. Additional Therapeutic, Prophylactic or Diagnostic Agents

In some embodiments, sterile de-cellularized human amniotic fluid(D-HAF) is administered in combination with one or more additionaltherapeutic, diagnostic, and/or prophylactic agents to alleviate pain,facilitate healing, and/or reduce or inhibit scarring. In someembodiments, the composition may contain one or more additionalcompounds to relieve symptoms such as inflammation. Non-limitingexamples include antimicrobial agents, analgesics, local anesthetics,anti-inflammatory agents, anti-oxidants, immunosuppressants,anti-allergenic agents, enzyme cofactors, essential nutrients and growthfactors. Suitable therapeutic agents include but are not limited toantibiotics, cytokines, and growth factors such as fibroblast growthfactor, hepatocyte growth factor, platelet-derived growth factor,vascular endothelial cell growth factor, and insulin-like growth factor.In some embodiments, the one or more agents are hyaluronic acid, orglycosaminoglycans.

The active agents can be a small molecule active agent or a biomolecule,such as a peptide or protein, sugar or polysaccharide, lipid, nucleicacid or combination thereof. Suitable small molecule active agentsinclude organic and organometallic compounds. In some instances, thesmall molecule active agent has a molecular weight of less than about2000 g/mol, more preferably less than about 1500 g/mol, most preferablyless than about 1200 g/mol. The small molecule active agent can be ahydrophilic, hydrophobic, or amphiphilic compound.

In some cases, one or more additional agents may be dispersed,dissolved, or suspended in the formulation, the sterile decellularizedamniotic fluid or pharmaceutically acceptable carrier.

The amount of a therapeutic generally depends on the tissue to betreated. Specific dosages can be readily determined by those of skill inthe art. See Ansel, Howard C. et al. Pharmaceutical Dosage Forms andDrug Delivery Systems (6th ed.) Williams and Wilkins, Malvern, Pa.(1995). Alternatively, the sterile de-cellularized human amniotic fluidcan be used in combination with cell delivery, for example, the deliveryof stem cells, pluripotent cells, somatic cells, or combinationsthereof.

In other embodiments, one or more therapeutic, prophylactic ordiagnostic agent is administered prior to, in conjunction with,subsequent to, or alternation with treatment with the sterilede-cellularized human amniotic fluid.

The therapeutic, prophylactic or diagnostic agent may be present in itsneutral form, or in the form of a pharmaceutically acceptable salt. Insome cases, it may be desirable to prepare a formulation containing asalt of an agent due to one or more of the salt's advantageous physicalproperties, such as enhanced stability or a desirable solubility ordissolution profile.

Generally, pharmaceutically acceptable salts can be prepared by reactionof the free acid or base forms of an active agent with a stoichiometricamount of the appropriate base or acid in water or in an organicsolvent, or in a mixture of the two; generally, non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Pharmaceutically acceptable salts include salts of an activeagent derived from inorganic acids, organic acids, alkali metal salts,and alkaline earth metal salts as well as salts formed by reaction ofthe drug with a suitable organic ligand (e.g., quaternary ammoniumsalts). Lists of suitable salts are found, for example, in Remington'sPharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000, p. 704.

In some cases, the additional agent is a diagnostic agent imaging orotherwise assessing the site of application. Exemplary diagnostic agentsinclude paramagnetic molecules, fluorescent compounds, magneticmolecules, and radionuclides, x-ray imaging agents, and contrast media.

In certain embodiments, the pharmaceutical composition contains one ormore local anesthetics. Representative local anesthetics includetetracaine, lidocaine, amethocaine, proparacaine, lignocaine, andbupivacaine. In some cases, one or more additional agents, such as ahyaluronidase enzyme, is also added to the formulation to accelerate andimproves dispersal of the local anesthetic.

In some embodiments, sterile de-cellularized human amniotic fluid areused in combination with one or more antimicrobial agents. Anantimicrobial agent is a substance that kills or inhibits the growth ofmicrobes such as bacteria, fungi, viruses, or parasites. Antimicrobialagents include antiviral agents, antibacterial agents, antiparasiticagents, and anti-fungal agents. Representative antiviral agents includeganciclovir and acyclovir. Representative antibiotic agents includeaminoglycosides such as streptomycin, amikacin, gentamicin, andtobramycin, ansamycins such as geldanamycin and herbimycin,carbacephems, carbapenems, cephalosporins, glycopeptides such asvancomycin, teicoplanin, and telavancin, lincosamides, lipopeptides suchas daptomycin, macrolides such as azithromycin, clarithromycin,dirithromycin, and erythromycin, monobactams, nitrofurans, penicillins,polypeptides such as bacitracin, colistin and polymyxin B, quinolones,sulfonamides, and tetracyclines.

Other exemplary antimicrobial agents include iodine, silver compounds,moxifloxacin, ciprofloxacin, levofloxacin, cefazolin, tigecycline,gentamycin, ceftazidime, ofloxacin, gatifloxacin, amphotericin,voriconazole, natamycin.

In some embodiments, sterile de-cellularized human amniotic fluid isadministered in combination with one or more local anesthetics. A localanesthetic is a substance that causes reversible local anesthesia andhas the effect of loss of the sensation of pain. Non-limiting examplesof local anesthetics include ambucaine, amolanone, amylocaine,benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine,butacaine, butamben, butanilicaine, butethamine, butoxycaine,carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine,dibucaine, dimethysoquin, dimethocaine, diperodon, dycyclonine,ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine,euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine,isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine,mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine,naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine,phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine,prilocaine, procaine, propanocaine, proparacaine, propipocaine,propoxycaine, psuedococaine, pyrrocaine, ropivacaine, salicyl alcohol,tetracaine, tolycaine, trimecaine, zolamine, and any combinationthereof. In other aspects of this embodiment, the sterile DHAFcomposition includes an anesthetic agent in an amount of, e.g., about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8% about 0.9%, about 1.0%, about 2.0%, about 3.0%, about4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, orabout 10% by weight of the total composition. The concentration of localanesthetics in the compositions can be therapeutically effective meaningthe concentration is adequate to provide a therapeutic benefit withoutinflicting harm to the patient.

In some embodiments, sterile de-cellularized human amniotic fluid isused in combination with one or more anti-inflammatory agents.Anti-inflammatory agents reduce inflammation and include steroidal andnon-steroidal drugs. Suitable steroidal active agents includeglucocorticoids, progestins, mineralocorticoids, and corticosteroids.Other exemplary anti-inflammatory agents include triamcinoloneacetonide, fluocinolone acetonide, prednisolone, dexamethasone,loteprendol, fluorometholone, ibuprofen, aspirin, and naproxen.Exemplary immune-modulating drugs include cyclosporine, tacrolimus andrapamycin. Exemplary non-steroidal anti-inflammatory drug includeketorolac, nepafenac, and diclofenac.

In some embodiments, anti-inflammatory agents are anti-inflammatorycytokines. Exemplary cytokines are IL-10, TGF- and IL-35.Anti-inflammatory cytokines in the context of biomaterial implant, andtissue grafts are cytokine that induce an anti-inflammatory immuneenvironment or suppress inflammatory immune environment. Activation ofregulatory T cells, Tregs, is involved in the prevention of rejection,the induction and maintenance of peripheral tolerance of the allograft.Th17 cells are a subset of T helper cells which is characterized by theproduction of IL-17. Th17 cells have been suggested to play a role inallograft rejection. In some embodiments, cytokines to be added to thesterile DHAF compositions are those that induce Tregs activation (e.g.,IL-25) and suppress Th17 activation (e.g., IL-10) for minimizingrejection.

In some embodiments, sterile de-cellularized human amniotic fluid isadministered in combination with one or more growth factors. Growthfactor, also known as a cytokine, refers to a protein capable ofstimulating cellular growth, proliferation, and/or cellulardifferentiation. Non-limiting examples of growth factors includetransforming growth factor beta (TGF-), transforming growth factor alpha(TGF-a), granulocyte-colony stimulating factor (GCSF),granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growthfactor (NGF), neurotrophins, platelet-derived growth factor (PDGF),erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF8), growthdifferentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGFor FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epidermalgrowth factor (EGF), vascular endothelial growth factor (VEGF)andhepatocyte growth factor (HGF).

In some embodiments, the sterile de-cellularized human amniotic fluidcomposition is administered in combination with one or more enzymecofactors, and/or one or more essential nutrients. Exemplary cofactorsinclude vitamin C, biotin, vitamin E, and vitamin K. Exemplary essentialnutrients are amino acids, fatty acids, etc.

In some embodiments, the sterile de-cellularized human amniotic fluidcomposition further comprises at least one eukaryotic cell type notpresent in the original amniotic fluid. Some exemplary eukaryotic celltypes include stem cells, mesenchymal stem cells, immune cells such as Tlymphocytes, B lymphocytes, natural killer cells, macrophages, dendriticcells, or combinations thereof. In some embodiments, the cells used arecells that dampen inflammation response such as regulatory T cells.

C. Formulations

In some embodiments, the sterile D-HAF compositions are packaged, forexample, into sterile dosage units which can be stored and distributedfor use by attending physicians. These lyophilized or fluid formulationscan be in the form of sterile packaged syringes for injection, dropperbottles, tubes or vials of solution. The dosages for the injectablestypically will be 0.1cc, 0.25 cc, 0.5cc, 1.0 cc, 2cc, 5cc, 10cc, and20cc. The injectables can be administered at the site of injury. In oneembodiment, the formulation is sprayed onto, soaked into, or powderdispersed onto the implant or prosthetic. The efficacy is determined byphysician evaluations, patient self-evaluations, and Quality of lifeevaluations.

In the preferred embodiment, the amniotic fluid is administered withoutany other excipient.

However, the sterile amniotic fluid formulation can be administered inconcentrated form, diluted with sterile water or buffer, formulated as asolution or suspension. It can include additional therapeutic,prophylactic or diagnostic agent, either in the solution or suspension,or as particles (nanoparticles, liposomes, microparticles) or implants.

Representative excipients include solvents, diluents, pH modifyingagents, preservatives, antioxidants, suspending agents, wetting agents,viscosity modifiers, tonicity agents, stabilizing agents, andcombinations thereof. Suitable pharmaceutically acceptable excipientsare preferably selected from materials which are generally recognized assafe (GRAS) and may be administered to an individual without causingundesirable biological side effects or unwanted interactions.

1. Solutions and Suspension

Numerous formulations are known and available. Solutions can be thesterile filtered amniotic fluid, concentrated or diluted with water,buffered saline, or an equivalent, or emulsified with lipid or oil.Emulsions are generally dispersions of oily droplets in an aqueousphase. There should be no evidence of breaking or coalescence.Suspensions contain solid particles dispersed in a liquid vehicle; theymust be homogeneous when shaken gently and remain sufficiently dispersedto enable the correct dose to be removed from the container. A sedimentmay occur, but this should disperse readily when the container isshaken, and the size of the dispersed particles should be controlled.The active ingredient and any other suspended material must be reducedto a particle size small enough to prevent irritation and damage to thecornea. They may contain suitable additives, such as antimicrobialagents, antioxidants, and stabilizing agents.

When the solution is dispensed in a multidose container that is to beused over a period of time longer than 24 hours, a preservative must beadded to ensure microbiologic safety over the period of use.

Ideally, the pH of should be physiological, for example, pH 7.4.However, the decision to add a buffering agent should be based onstability considerations. The pH selected should be the optimum for bothstability of the active pharmaceutical ingredient and physiologicaltolerance. If a buffer system is used, it must not cause precipitationor deterioration of the active ingredient. The normal useful range is6.5 to 8.5, although lower pH may be used. The final pH of the solutionis often a compromise, because many drugs have limited solubility andstability at the desired pH of 7.4. Buffers or pH adjusting agents orvehicles can be added to adjust and stabilize the pH at a desired level.The buffers are included to minimize any change in pH during the storagelife of the drug; this can result from absorbed carbon dioxide from theair or from hydroxyl ions from a glass container. Changes in pH canaffect the solubility and stability of drugs; consequently, it isimportant to minimize fluctuations in pH. The buffer system should besufficient to maintain the pH throughout the expected shelf-life of theproduct. Low concentrations of buffer salts are used to prepare buffersof low buffer capacity.

The preparation of aqueous solutions requires careful consideration ofthe need for isotonicity, a certain buffering capacity, the desired pH,the addition of antimicrobial agents and/or antioxidants, the use ofviscosity-increasing agents, and the choice of appropriate packaging.Solutions are considered isotonic when the tonicity is equal to that ofa 0.9% solution of sodium chloride. Tissue can usually toleratesolutions equivalent to 0.5-2% of sodium chloride.

Solutions that are isotonic are preferred. An amount equivalent to 0.9%NaCl is ideal for comfort and should be used when possible. There aretimes when hypertonic solutions are necessary therapeutically, or whenthe addition of an auxiliary agent required for reasons of stabilitysupersedes the need for isotonicity. A hypotonic solution will requirethe addition of a substance (tonicity adjusting agent) to attain theproper tonicity range.

A widely used buffer solution is Sorensen's modified phosphate buffer.Sorensen's modified phosphate buffer is used for drugs needing pH valuesbetween the range of 6.5-8.0. This buffer uses two stock solutions, oneacidic containing NaH2PO4, and one basic containing Na₂HPO₄. Othersuitable buffers are well known by those skilled in the art such asacetate, borate, carbonate, citrate, and phosphate buffers.

In some instances, the formulation is distributed or packaged in aliquid form. Alternatively, formulations can be packed as a solid,obtained, for example by lyophilization of a suitable liquidformulation. The solid can be reconstituted with an appropriate carrieror diluent prior to administration.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more tonicity agents to adjust the isotonic range of theformulation. Suitable tonicity agents are well known in the art and someexamples include glycerin, mannitol, sorbitol, sodium chloride, andother electrolytes.

Solutions, suspensions, aerosols, sprays or emulsions may also containone or more preservatives to prevent bacterial contamination of theophthalmic preparations. Suitable preservatives are known in the art,and include polyhexamethylenebiguanidine (PHMB), benzalkonium chloride(BAK), stabilized oxychloro complexes (otherwise known as PURITE®),phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine,benzyl alcohol, parabens, thimerosal, and mixtures thereof.

Solutions, suspensions, or emulsions may also contain one or moreexcipients known art, such as dispersing agents, wetting agents, andsuspending agents.

D. Kits

In some embodiments, the compositions are provided in a kit.Formulations are prepared using a pharmaceutically acceptable “carrier”composed of materials that are considered safe and effective and may beadministered to an individual without causing undesirable biologicalside effects or unwanted interactions. These lyophilized or fluidformulations can be in the form of sterile packaged syringes forinjection, or tubes or jars of solution. The dosages for the injectableswill be 0.1cc, 0.25 cc, 0.5cc, 1.0 cc, 2cc, 5cc, 10cc, and 20cc.Typically the sterile DHAF compositions will be in a single dose unit,or in a kit with a first containing with liquid to rehydrate the drycomponents in a second component. These may include components foradministration, such as syringe and an applicator such as a needle.

III. Methods of Administration

Methods of using sterile, de-cellularized human amniotic fluidcompositions for therapeutic, diagnostic, and prophylactic applications,especially application to joints, tendons, ligaments, cartilage or otherconnective tissue are provided.

In certain embodiments, the compositions are administered to promotejoint or cartilage repair, and/or regeneration in a subject compared tocompositions and methods that do not incorporate the use of the steriledecellularized amniotic fluid. In more specific embodiments, thecompositions and methods promote integration of implants or graftedtissues in a subject into the implanted/grafted region, and results in abetter “take” and reduced rejection of the implants or grafted tissues.The compositions can be administered to tissues, joints, ligaments,tendons, cartilage, or connective tissue directly, or indirectly via adressing, cement or glue or hydrogel for tissue repair.

The formulations can be administered to any mammal, including humans,livestock (horses, sheep, cattle, and pigs) as well as domestic animals(dogs and cats being the most common). The decellularized amniotic fluidis administered in a dosing regimen and for a period of time effectiveto provide healing or repair or regeneration.

The DHAF composition may be retained at the site of application for anextended period of time. For example, after administration, one or moretherapeutic agent in the DHAF composition remains at the site ofapplication for at least 6 hours, at least 12 hours, at least 1 day, atleast 2 days, at least 3 days, at least one week, at least 2 weeks, atleast 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, atleast 7 weeks, at least 2 months, at least 3 months, at least 6 months,at least 9 months, at least 1 year or more.

A. Disorders and Diseases to be Treated

The compositions and methods of use thereof are suitable for managing ortreating any pain, disease, disorder, degeneration or injury of a jointor associated soft tissue in human patients as well in animals.

A joint is where two or more bones come together, like the knee, hip,elbow, or shoulder. Joints can be damaged by many types of injuries ordiseases, including arthritis (inflammation of a joint), bursitis(inflammation of a fluid-filled sac that cushions the joint), anddislocations (injuries that force the ends of the bones out ofposition). Some exemplary joint disorders are effusion of unknown originor suspected infection, crystalloid arthropathies, synovitis, arthritisincluding, but not limited to, inflammatory arthritis, and advancedosteoarthritis, and others affect the joint membranes themselves. Othertypes of joint conditions can benefit from the use of the compositionsare those affect the tendons, cartilage, bursae, ganglion cysts, triggerpoints, neuromas, entrapment syndromes, fasciitis, and fluid within thejoint (synovial fluid) such as bursitis, tendinitis, and tenosynovitis.In some embodiments, the compositions and methods of use thereof are fortreating, and/or alleviating one or more symptoms of arthritis,bursitis, dislocations, tendinitis, and tenosynovitis.

Any joint condition may be treated, including joints with pain,arthritis, osteochondrosis, fibrous dysplasia, bone tumor, degeneration,cartilage wear or tear, and joints that have been fractured. In someembodiments, the formulation is administered to bones that have hadprosthetic implants, pins, screws or plates attached or implanted intothem, to promote healing, repair, and/or to reduce inflammation. In someembodiments, injections of the disclosed are administered to promoteresurfacing, repair, and/or regeneration of cartilage. In someembodiments, intraarticular injection of the formulation to an arthriticjoint space of a mammalian host results in regenerating hyalinecartilage. After the injections of the formulation, the percentage ofhyaline cartilages repaired, and/or regenerated is about 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the initiallydamaged, worn cartilage, in volume.

Methods of using the D-HAF to stimulate cartilage regeneration aredescribed. In some embodiments, the methods and compositions areeffective in repairing and/or regenerating surface cartilage such asarticular cartilage. In further embodiments, the formulation isadministered in an amount effective to regenerate articular cartilage inthe femoral, tibial, and/or patellar articular surfaces to about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, ormore than 300% of the damaged or worn cartilage present at the time oftreatment, measured by cartilage thickness.

Methods of using the D-HAF to stimulate bone growth in a subject in needthereof are also described. In some embodiments, the formulation isadministered in an amount effective to stimulate bone growth for healingof bone fractures and/or bone grafting.

Methods of using the compositions to reduce or prevent bone loss in asubject are provided. In some embodiments, the formulation isadministered in an amount effective to reduce and/or prevent bone loss.In some embodiments, the subject is in need of treatment for osteopeniaor osteoporosis. In some embodiments, the compositions are administeredto a subject in an effective amount to reduce or inhibit boneresorption, and/or increase bone formation in a subject in need thereof.In some embodiments, the compositions are effective to reduce the rateof bone resorption by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, or more than 200%. In some embodiments, the compositionsincrease the rate of calcium deposition by osteoblasts into bone matrixby 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or morethan 200%.

Methods of using the D-HAF to increase the Wnt signaling pathway aredescribed. Typically, the compositions are effective in increasing theactivity and/or quantity of one or more members associated with the Wntsignaling pathway. In some embodiments, the compositions lead to directand/or indirect increase in the activity, protein level, and/ortranscript level of one or more members of the Wnt signaling pathway. Insome embodiments, the compositions are administered to a subject todeliver an effective amount Wnt4 to activate the Wnt signaling pathwayfor tissue regeneration. In some embodiments, the Wnt signaling pathwayto be modulated is the canonical Wnt/-catenin signaling pathway,including Wnt3A, Wnt receptor frizzled 1, Disheveled, Axin, Frat-I,and/or -catenin. In some embodiments, the Wnt signaling pathway to bemodulated is the non-canonical Wnt signaling pathway.

In some embodiments, the formulation is administered to assist inconnective tissue repair. The connective tissue includes, but is notlimited to, cartilage, ligament, tendon, a bone, and a synovium of amammalian host. In some embodiments, the formulation is administered toassist in repair of tom or strained ligaments or tendons. In oneembodiment, the formulation is administered by injection to joints,bursae, tendon sheaths, or soft tissues at/near the site of injury. Inanother embodiment, the formulation is sprayed onto, soaked into, orpowder dispersed onto the implant or prosthetic. This can includematrices, implants and sutures.

B. Treating Joint Disorders in Animals

The formulations may be administered to animals as well as to humans totreat or alleviate pain, disease, disorder, degeneration or injury of ajoint or associated soft tissue including tendons, ligaments. Preferredanimals include dogs, cats, horses, as well as other animals that can betreated including livestock (cattle, sheep, swine, goats). In someembodiments, the animals in need of treatment have one or more jointconditions including arthritis, bursitis, tendinitis, and tenosynovitis.

The compositions and methods are useful for managing or treating jointdisorders in horses. The formulation can be administeredprophylactically or therapeutically. In many cases, regularadministration is used to prevent further damage. Examples of horsesrequiring prophylactic treatment include racehorses, barrel, reining,and cutting horses, jumpers and dressage horses. Treatment forprophylaxis is typically done using a slightly lower dose and injectedinto the affected or at risk joints. A series of injections may beadministered into or at the joint, tendons or ligaments for chronicinjury.

The most common joint issues in dogs are hip dysplasia, elbow dysplasiaand displaced patellas, as well as arthritis due to age or trauma. As inthe case of the horses, treatment may be administered prophylactically,on a regular basis for chronic injury or escalated dosage for acuteinjury.

In all cases the formulation may be administered directly as in the caseof surgery for an injury or implantation of a prosthetic, pins orscrews.

All joint diseases that affect large animals produce some degree ofinflammation, often with accompanying swelling, pain, redness or heat.Important arthritic conditions include traumatic arthritis,osteochondritis dissecans, subchondral cystic lesions, septic(infectious) arthritis, and osteoarthritis. In some embodiments, thecompositions and methods of use thereof are for treating, alleviatingone or more symptoms of traumatic arthritis, osteochondritis dissecans,subchondral cystic lesions, septic (infectious) arthritis, orosteoarthritis in animals such as horses and dogs.

In some embodiments, the compositions and methods of use thereof are fortreating, and/or alleviating one or more symptoms of hip dysplasia,elbow dysplasia, or panosteitis in animals such as dogs. In someembodiments, the compositions and methods of use thereof are fortreating, and/or alleviating one or more symptoms of disorders of thehip such as coxitis, dislocations, fractures, and bursitis in animalssuch as horses and dogs.

Common in horses, bursitis is an inflammatory reaction within a bursathat can range from mild inflammation to infection. True bursitisinvolves inflammation of a natural bursa, for example, fistulouswithers, or poll evil. Fistulous withers is a condition in which thebursa in a horse's withers region (the ridge between the shoulder bones)becomes infected and inflamed. Poll evilis a virtually identicalcondition, except that the location of infection is the bursa behind thehorse's ears in the area known as the poll. In each instance, a rupturedbursal sac creates an opening that makes the bursa susceptible toinfection. In contrast, acquired bursitis is the development orinflammation of a bursa where none previously existed, as with cappedelbow or hock. In capped elbow and hock, bursas beneath the hock andelbow can become inflamed if a horse suffers trauma from falling in orkicking its stall, from lying on poorly bedded hard floors, riding atrailer tailgate, wearing iron shoes that project beyond the heels, orfrom leaning or reclining for long periods of time. In some embodiments,the compositions and methods of use thereof are for treating, and/oralleviating one or more symptoms of bursitis in animals such as horses.

Tendinitis is the inflammation of a tendon. Tendon inflammation may beshort-term (acute tendinitis) or build up over a period of time (chronictendinitis). The condition is most common among horses that do fastwork, such as racehorses. Tenosynovitis is an inflammation of thesynovial membrane and usually the outer fibrous covering of the tendon.Possible causes include a response to traumatic injury or infection.When seen in young animals where the cause is uncertain, the conditionis called idiopathic synovitis. In some embodiments, the compositionsand methods of use thereof are for treating, and/or alleviating one ormore symptoms of tendinitis, tenosynovitis, or idiopathic synovitis inanimals such as horses.

The compositions and methods of managing or treating joint disorders indogs and cats are also disclosed. Some exemplary joint disorders in dogsand cats include arthritis, bursitis, tendinitis, and tenosynovitis. Insome embodiments, the compositions and methods of use thereof are fortreating, and/or alleviating one or more symptoms of joint disorderssuch as arthritis, bursitis, tendinitis, and tenosynovitis in dogs andcats. The most common signs of joint disease in dogs and cats includestiffness, limping, or favoring a limb—particularly after sleep orresting, inability to rise, reluctance to jump or even climb stairs, andnoticeable pain.

C. Dosages and Dosing Regimen

Dosage and dosing regimens are dependent on the severity of thecondition of joint and/or soft tissue to be treated, and are known tothose skilled in the art. The formulation is tailored to the individualsubject, the nature of the condition to be treated in the subject, andgenerally, the judgment of the attending practitioner. In yet anotherembodiment, the formulation is any amount of about 0.1cc to 20 cc.

The standard protocol is a series of three injections that can be donedaily, every other day, once a week, once a month, or quarterly,depending on the indication and on the severity of the injury orinflammation. In preferred embodiments, the larger hip and shoulderjoints get 1.9 cc of PDA Human Amniotic Fluid, and the smaller jointsget 0.5 cc, and the fingers and feet get 0.25 cc.

A filled ampoule contains a formulation of D-HAF. This is generally in apharmaceutically acceptable carrier and buffered for use in human oranimal subjects to a pH of about 3.5-10.0. The formulations of theexamples are buffered to about pH 6.0-7.5. The formulations are free ofpreservative, which is an advantage as some preservatives can beassociated with side effects such as synovitis. Water or saline solutionis used to provide the carrier.

One or more tonicity adjusting agents may be added to provide thedesired ionic strength. Tonicity-adjusting agents for use include thosewhich display no or only negligible pharmacological activity afteradministration. Both inorganic and organic tonicity adjusting agents maybe used. Compositions can also include excipients and/or additives.Examples of these are surfactants, stabilizers, complexing agents,antioxidants, or preservatives which prolong the duration of use of thefinished pharmaceutical formulation, flavorings, vitamins, or otheradditives known in the art. Complexing agents include, but are notlimited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof,such as the disodium salt, citric acid, nitrilotriacetic acid and thesalts thereof. In one embodiment, the complexing agent is EDTA.Preservatives include, but are not limited to, those that protect thesolution from contamination with pathogenic particles, includingbenzalkonium chloride or benzoic acid, or benzoates such as sodiumbenzoate. Antioxidants include, but are not limited to, vitamins,provitamins, ascorbic acid, vitamin E or salts or esters thereof.

In some embodiments, lyophilized D-HAF formulations are preferred. Insome embodiments, the lyophilized D-HAF is reconstituted by adding theinitial volume of water. In other embodiments, the formulation isfurther diluted to from about 1% to about 99% of the reconstitutedD-HAF. The refrigerated formulation is readily diluted to from about 1%to about 99% of the original D-HAF to a desired concentration forapplications.

In other embodiments, the final formulation is prepared as a much moreconcentrated solution depending on the need of application. In oneembodiment, the lyophilized D-HAF is reconstituted by adding half of theinitial volume of water to achieve twice as concentration solutions ofall amniotic factors. In a further embodiment, the lyophilized D-HAF isreconstituted by adding 10% of the initial volume of water to achieve10-fold more concentrated solutions of the amniotic factors forapplication. In some embodiments, the refrigerated D-HAF can be used toreconstitute the lyophilized D-HAF in order to obtain a moreconcentrated solution.

The D-HAF formulations can be administered as frequently as necessaryall appropriate. The frequency generally depends on the severity of thejoint/soft tissue condition, and the responsiveness of the targettissues to the treatment with D-HAF formulations. In some embodiments,the D-HAF formulations are administered on one-a-week basis. In otherembodiments, the D-HAF formulations are administered on one-a-monthbasis. In some embodiments, the administration routine can change basedon the practitioners assessment of the patient after the priortreatment.

Since there is no toxicity known to be associated with the formulation,it can be injected as often as the physician chooses, unlike steroidsthat can only be injected infrequently, typically two to three times ayear.

The present invention will be further understood by reference to thefollowing non-limiting examples.

D. Controls

The effect of the composition can be compared to a control. Suitablecontrols are known in the art and include, for example, an untreatedsubject or another part of the same individual (example left kneetreated, right knee untreated) or a placebo-treated subject. In someembodiments, an untreated control subject suffers from, the same diseaseor condition as the treated subject e.g. osteoarthritis, osteoporosis,preferably having a similar degree of severity.

In some embodiments, a suitable control is an area having the same bonedensity as the area being treated on the same subject. For example, apatient with osteoporosis on both wrists can treat one wrist locallywith the compositions, whilst use the other as a control.

EXAMPLES

The present invention will be further understood by reference to thefollowing non-limiting examples. The examples showing preparation ofhuman amniotic formulation are from US20150025366.

Example 1 Preparation of Human Amniotic Formulation

Materials and Methods

Human amniotic fluid is collected from selected caesarean sections,which make aspiration of the amniotic fluid in clean condition possible.Then the amniotic fluid is stored in refrigerated condition at 2° C. to6° C. before the clarification and filtration process. The amnioticfluid is centrifuged at 5,000 to10,000 rpm for 30 minutes to 1 hour in50 mL to 250 mL swing out buckets. The supernatant is collected. Whencollecting the supernatant, it is important to avoid detaching oraspirating insoluble components possibly coming from the pellet or fromthe fatty overlayer. If the supernatant still contains residualinsoluble components, it may be pre-filtered with 5 to 10μ celluloseester capsule pre-filters without TRITON® surfactant to avoidcontamination in the filtration process. The liquid phase is collectedand filtered with poly ether sulfone 1.0μ capsule filters and the liquidis collected. The liquid is then filtered with poly ether sulfone 0.2μcapsule filter. The filtrate is transferred to vials and sealed withstoppers aseptically. Four samples from the final filtrate are taken totest whether the sterile filtered human amniotic fluid retains growthfactors, such as human growth hormone, transforming growth factor beta1, vascular endothelial growth factor, epidermal growth factor,transforming growth factor beta 3.

The amniotic fluid from the final filtration is aseptically transferredto syringes or vials, then kept in a deep freezer at about −80° C. toabout −20° C. for long term storage. The sterile amniotic fluid is driedin the vial via lyophilization in a built-in a sterile environment. Thelyophilisate derived from the amniotic fluid is reconstituted withsterile water before its injection or topical administration. Thelyophilisate can be stored at from +4° C. to about +25° C. (roomtemperature). All of this operation may be carried out in sterilecondition and does not need additional sterilization methods such as afinal irradiation.

If needed, the lyophilisate derived from amniotic fluid throughlyophilization may be irradiated by e-beam irradiation or gamma rayirradiation to add another guarantee for the final sterility of thepowder. Irradiation of a lyophilisate is much less denaturing forproteins and peptides than irradiating aqueous solutions, because theabsence of water considerably reduces the production of reactivesuperoxide anions and their diffusion during irradiation. Suchsuperoxide anions are the main cause of splitting peptide bonds andchemically modifying amino acids of protein and peptides. Afterlyophilization, the amniotic fluid is reconstituted by adding theinitial volume of water. After gentle homogenization, the powder isquickly dissolved in about one minute.

Results

The results show retention of growth factors. The concentration of thegrowth factors in the sterile filtered amniotic fluid is from about 30pg/mL to about 2500 pg/mL. Except the vascular endothelial growth factorin sample 2, the concentrations of all the factors in the four samplesare in the range of 30-150 pg/mL. Although part of growthdifferentiation factor 11 is lost in centrifugation and filtration, thefinal sterile filtered amniotic fluid still retains about 17% to 29% ofgrowth differentiation factor from the raw human amniotic fluid.

Example 2 Administration to Human Patients with Joint Disease orDisorders

A patient with Spasmodic Torticollis, an extremely painful chronicneurological disorder, was treated by injection of the formulation totreat a bilateral rotator cuff injury.

The formulation significantly reduced pain and increased the rate ofhealing.

Example 3 Treatment of Severe Knee Pain

A 68 year old male with bi-lateral severe knee pain indicated the painwas a 9 or 10 on a pain scale from 0 to 10, with 0 being no pain and 10being the worst pain. The pain also radiated down the legs from time totime. Flexibility was greatly hampered, with difficulty in putting onshoes.

The individual was treated with a series of platelet rich plasma (“PRP”)and Hylauronic Injections, neither working very well. Pain was notlessened, and mobility was not greatly improved.

After one injection with the human amniotic fluid into both knees, therewas pain relief. Within a few days pain went from a 9 to 10 to a 2 to 3and has improved since then. Mobility was substantially increased.

Example 4 Treatment of Foot

A patient was treated by injection following foot surgery to insertscrews and realign the foot bones. Swelling was significantly reduced.Pain was reduced from a 7-8 to a 3-4.

Example 5 Treatment of Neck Injury/Pain

Individual had a cheerleading injury in High School. No problem untilentering her forties. MRI showed severe disc degeneration between L3 andL4. The pain was excruciating. Recommended treatment was steroidinjections and Radio Frequency Ablation (“RF”) and ultimately, surgery.Over the next five years the patient had steroid injections that did notwork, and two RF treatments 18 months apart, that did help a little.

Patient was this injected with amniotic fluid formulation and again twoweeks later and again two weeks later.

With five hours after the second injection, pain went from an 8 on thepain scale to a 4. After third injection, pain is at a 2 and verymanageable without medication.

Example 6 Treatment of Osteoarthritis

Patient is stooped, with some finger deformation, and shoulder and kneeproblems. Patient has had a total right knee replacement and isconsidering an operation to his left knee. He has not had any surgerieson his shoulder.

He received one injection to his left shoulder: a posterior approach.Prior to treatment patient was able to raise his arm until it waslateral to the ground, with no pain. Any higher caused pain, whichbecame excruciating by the time arm was perpendicular to the ground. Aprevious steroid injection provided no pain relief or increase inmobility.

After treatment there is full mobility of arm with no pain.

Example 7 Treatment of Osteoarthritis with Cartilage Regeneration

A 62-year old male with degenerative osteoarthritis of the left knee wastreated. After treatment, Multiplanar, multisequence Magnetic ResonanceImaging of the left knee was performed without intravenous contrast.

Recommended sequences from the International Society of Cartilage Repairwere performed, which included the addition of a 3-D Tl weighted GREsequence in addition to standard departmental knee MRI protocol.

MRI findings indicated that there had been interval rupture of theanterior cruciate ligament (ACL) but the PCL, MCL, and LCL are intact.The quadriceps and patellar tendons were intact. Complex tearing of themedial meniscus was noted but the lateral meniscus remains intact. Therewas no evidence of muscular tear, muscle strain, or muscle atrophy.There was popliteus bursitis, less extensive than the prior study. Therewas a small loose body within the popliteus bursa measuring 5×8 mm;there was a small joint effusion, which was similarly decreased from theprior study; there was debris within the joint effusion, which couldrepresent synovitis; and there was still cartilage loss in the medialcompartment with subchondral marrow signal changes. There was completeregeneration of an area of focal cartilage loss along the anterioraspect of the lateral femoral condyle and restoration of normalsubchondral marrow signal. The lateral compartment articular cartilagewas intact. There had been no progression of the loss of articularcartilage along the medial patellar face. The remaining articularcartilage in the patellofemoral compartment was intact. There weretricompartmental osteophytes, most pronounced in the medial compartment.There were small marginal osteophytes arising from the lateral andpatellofemoral compartments. There was no evidence of acute fracture,dislocation, or destructive bony lesion.

Radiologist interpretation of the MRI results include: 1) Completeregeneration of an area of focal cartilage loss along the anterioraspect of lateral femoral condyle and restoration of normal subchondralmarrow signal. The lateral compartment articular cartilage wascompletely intact. There had been no progression of loss of thearticular cartilage along the medial patellar facet. There waspersistent cartilage loss in the medial compartment with subchondralmarrow signal changes. 2) Interval rupture of the ACL. Complex tearingof the medial meniscus was again observed. 3) Interval decrease in sizeof anterior knee effusion. Only a tiny knee effusion remained. Intervaldecrease in size of fluid within the popliteal bursa. A small loose bodywas within the popliteal bursa.

The patient had pain of 8 (on the scale of 1 to 10, 10 being the worse)and was barely able to walk. The patient had D-HAF (or Sterile D-MAPPSderived Liquid Allograft) injections into his left knee. Within a year,the patient reported that he was pain-free and was able to walk and runwithout pain.

Example 8 Cartilage Regeneration

A 59-year old male who was an avid golfer was injected with D-HAF.Significant cartilage regeneration was observed using Magnetic resonanceimaging (MRI).

MRI images were obtained prior to the treatment; and post-treatmentimages were taken a month later. The MRI findings indicate increasingthickness of the articular cartilage in the femoral, tibial and patellararticular surfaces. Based on the MRI images taken prior to thetreatment, the thickness of femoral articular surface was about 1.09 mm;

and the thickness of tibial articular surface was about 2.35 mm. Thepost-treatment MRI images show that the thickness of femoral articularsurface was about 4.78 mm; and the thickness of tibial articular surfacewas about 3.70 mm. Thus, the increase in the thickness of femoralarticular surface was about 3.75 mm, and the increase of tibialarticular surface was about 1.35 mm. The combined increase in thearticular surfaces in the knee was about 5.1 mm.

Example 9 Improving Chronic Knee Pain

22 patients with chronic knee pain were treated with injectable D-HAF.Their pain levels were measured prior to the treatment, and weremonitored at 3-month, 6-month, and 12-month intervals post treatment(Table 1). Their average pain levels throughout the 12-month period weresummarized in FIG. 2. Three months after treatment, there was an 82.2%improvement in their pain levels; 6 months after treatment, there was an87.5% improvement in their pain levels; and maintained at a similarlevel after 12 months with an 82.2% improvement.

TABLE 1 Summary of improvement in patients with chronic pain pre- andpost- treatment. Distribution in 3 months 6 months 12 months No. of Eachpost post post Pain Level Patients Level treatment treatment treatment10  6 27% 1.667 1.000 0.000  9  5 23% 1.600 0.800 1.000  8  5 23% 0.0000.000 0.000  7  4 18% 3.750 3.333 8.000  6  2  9% 0.000 0.000 N/A TotalNo. 22 22 19 6 of Patients

Example 10 Protein Quantification of Wnt4 in the Sterile D-HAF

The Wnt signaling pathway plays a central role in bone development andhomeostasis. (McClung M R et al., N Engl J Med. 370(5):412-20 (2014);Minear Set al., Sci Transl Med.2(29):29ra30 (2010); Jing Wet al.,Biomaterials. 47:29-40 (2015)).

The protein level of Wnt4 in the D-HAF was evaluated. With a range ofconcentrations of Wnt-4 protein, a standard curve was first generated(table 1, FIGS. 3A & 3B). The D-HAF sample was then tested for theprotein quantity of Wnt-4 (FIG. 3C). The estimated concentration ofWnt-4 in D-HAF was about 5000 pg/ml.

TABLE 2 Different concentrations of Wnt-4 for generating a standardcurve. Concentration 0 55 165 494 1481 444 13333 40000 (pg/ml)Signal-BKG 0 20 29 245 742 1893 7032 15473 Log 1.74 2.22 2.69 3.17 3.654.12 4.60 (Concentration) Log (Signal- 1.30 1.46 2.39 2.87 3.28 3.854.19 BKG)

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed:
 1. A method of promoting repair and/or regeneration ofcartilage in a subject comprising administering to or adjacent to a sitein need thereof an effective amount of a sterile, de-cellularized, humanamniotic fluid (D-HAF).
 2. The method of claim 1 wherein D-HAF is devoidof amniotic stem cells, elements of micronized membrane, and chorionparticles.
 3. The method of claim 1 wherein D-HAF comprises at least 300human growth factors.
 4. The method of claim 1, wherein the site in needthereof is a joint.
 5. The method of claim 1, wherein the cartilage istibial cartilage.
 6. The method of claim 1, wherein D-HAF isadministered in an amount effective to regenerate articular cartilage inthe femoral, tibial, and/or patellar articular surfaces.
 7. The methodof claim 4, wherein the joint has pain, arthritis, osteochondrosis,fibrous dysplasia, and/or a bone tumor.
 8. The method claim 7, whereinthe joint has degeneration, cartilage wear or tear, and/or joints thathave been fractured.
 9. The method of claim 7, including administeringD-HAF to bones with prosthetic implants, pins, or screws, the D-HAFadministered to promote healing, repair, and/or to reduce inflammation.10. A method for preventing bone loss or promoting bone formation in asubject in need thereof comprising administering to or adjacent to asite in need thereof an effective amount of a sterile de-cellularizedhuman amniotic fluid (D-HAF) devoid of amniotic stem cells, to reduceone or more symptoms of bone loss, prevent one or more symptoms of boneloss, promote bone formation, or combinations thereof, in the subjectrelative to an untreated control.
 11. The method of claim 10, whereinthe subject has osteopenia or osteoporosis.
 12. The method of claim 10,wherein the subject has a bone fracture or needs a spinal fusion. 13.The method of claim 10, wherein the subject is an animal.
 14. The methodof claim 13, wherein the animal comprises a dog, cat, horse, orlivestock.
 15. The method of claim 14, wherein the animal has arthritis,bursitis, tendinitis, and/or tenosynovitis.
 16. A composition comprisingD-HAF and one or more pharmaceutically acceptable excipients and/oradditives.
 17. The composition of claim 16, wherein D-HAF comprises aD-HAF formulation, the D-HAF formulation comprising a lyophilizedformulation or fluid formulation.
 18. The composition of claim 17,wherein the lyophilized formulation comprises a lyophilisate.
 19. Thecomposition of claim 18, wherein the lyophilisate is stored in arefrigerated condition at +2° C. to about +25° C.
 20. The composition ofclaim 16, wherein the process by which D-HAF is prepared comprises: (a)collecting human amniotic fluid from a caesarean section to produce asample of raw amniotic fluid; (b) centrifuging the sample of rawamniotic fluid at 5,000 to 10,000 rpm for 30 minutes to 1 hour; and (c)collecting the supernatant.
 21. The process of claim 20, wherein thefinal filtrate is stored in frozen condition at about −20° C. to about−80° C.